P450 Cyptide Synthase KwwB Catalyzes Trp-C5-Trp-N1 Cross-Linking and Accepts Diverse Precursor Peptides

Cytochrome P450 enzymes install C-C, C-N, or C-O cross-links on the precursor peptides of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we bioinformatically mapped P450 enzymes based on the known families. Through functional studies in Escherichia coli, the newly identified P450 cyptide synthase KwwB from Kitasatospora sp. GAS204B was found to catalyze the formation of a cross-link between Trp-C5 and Trp-N1 at the WxW motif. The substrate tolerance of the Trp-C5-Trp-N1 cross-linked P450 enzymes has not been determined. This work demonstrated that KwwB is a promiscuous enzyme capable of accepting a variety of substrates. This result provides an additional toolkit for cross-linked peptide modification.